Construction, expression, functional characterization and practical application of fusion protein SPA-BAPmut

Aim. The creation of genetically engineered fusion protein SPA-BAPmut and its application as a secondary immunoreagent in immunoassays. Methods. Gene cloning, PCR, electrophoresis, DNA sequencing, bacteria cells culturing, protein expression and purification, ELISA, Western-blotting were used. Results. The DNA sequences encoding Staphylococcus aureus protein A (SPA) and bacterial alkaline phosphatase with enhanced catalytic activity (BAPmut) were used for construction of gene encoding fusion protein SPA-BAPmut that was expressed in the high-productive Escherichia coli system and obtained in a soluble form. Cultivation conditions to provide a high-level expression of SPA-BAPmut (> 1 g/l) were determined. The target protein was obtained with purity more than 95 % using IMAX method. SPA-BAPmut is thermostable, and both parts of fusion protein (SPA and BAPmut) retain their IgG binding and alkaline phosphatase activity for a long time. SPA-BAPmut was used as a substitute of secondary antibodies in immunoassays. As little as 5 ng of the antigen could be detected in Western blotting and 1 g/ml of IgG in ELISA. Conclusions. The possibility of using SPA-BAPmut as universal secondary immunoreagent for different types of immunoassays was shown.Мета. Створення генно-інженерного злитого білка SPA-BAPmut та його застосування як вторинного імунореагенту в імунологічних тестах. Методи. Клонування генів, ПЛР, секвенування ДНК, культивування бактерій, електрофорез, синтез і очищення білків, ELISA, вестерн-блот. Результати. З використанням послідовновностей ДНК, що кодують білок А Staphylococcus aureus (SPA) і бактерійну лужну фосфатазу з покращеними каталітичними властивостями (BAPmut), сконструйовано ген злитого білка SPA-BAPmut та забезпечено його препаративне отримання у розчинній формі внаслідок синтезу в клітинах Escherichia coli. Визначено умови ферментації, за яких вихід SPA-ВAPmut становить приблизно 1 г/л культури E. coli. Із застосуванням методу металоафінної хроматографії одержано цільовий білок з чистотою понад 95 %. SPA-ВAPmut термостабільний, а обидва його компоненти (SPA і ВAPmut) зберігають імуноглобулінзв’язувальну і фосфатазну активність тривалий час. SPA-BAPmut дозволяє виявляти щонайменше 5 нг антигену та 1 мкг/мл антитіл. Висновки. Показано можливість використання SPA-ВAPmut як універсального вторинного імунореагенту в імунохімічних тестах.Цель. Создание генно-инженерного слитого белка SPA-BAPmut и eго использование как вторичного иммунореагента в иммунологических тестах. Методы. Клонирование генов, ПЛР, секвенирование ДНК, культивирование бактерий, электрофорез, биосинтез и очистка белков, ELISA, вестерн-блот. Результаты. С использованием последовательностей ДНК, кодирующих белок А Staphylo- coccus aureus (SPA) и бактериальную щелочную фосфатазу с улучшенными каталитическими свойствами (BAPmut), сконструирован ген слитого белка SPA-BAPmut и обеспечено его препаративное получение в растворимой форме вследствие синтеза в клетках Escherichia coli. Определены условия ферментации, при которых выход SPA-ВAPmut составляет около 1 г/л культуры E. coli. С применением метода металлоаффинной хроматографии целевой белок получен с чистотой более 95 %. SPA-ВAPmut термостабилен, а оба его компонента (SPA и ВAPmut) сохраняют иммуноглобулинсвязывающую и фосфатазную активность на протяжении длительного времени. SPA-BAPmut позволяет детектировать 5 нг антигена и 1 мкг/мл антител. Выводы. Показана возможность применения SPA-ВAPmut как универсального вторичного иммунореагента в иммунохимических тестах

The triple-pomeron regime and the structure function of the pomeron in the diffractive deep inelastic scattering at very small x

Misprints and numerical coefficients corrected, a bit of phenomenology and one figure added. The case for the linear evolution of the unitarized structure functions made stronger.Comment: KFA-IKP(Th)-1993-17, Landau-16/93, 46 pages, 14 figures upon request from N.Nikolaev, [email protected]

Nuclear shadowing in Glauber-Gribov theory with Q2-evolution

We consider deep inelastic scattering off nuclei in the Regge limit within the Glauber-Gribov model. Using unitarized parton distribution functions for the proton, we find sizeable shadowing effects on the nuclear total and longitudinal structure functions, $F_2^A$ and $F_L^A$, in the low-x limit. Extending a fan-diagram analysis for the large-mass region of coherent diffraction off nuclei to high Q2, we also find significant shadowing effects in this kinematical regime. Finally, we discuss shortcomings of our approach and possible extensions of the model to other kinematical regimes.Comment: 16 pages, 9 figure

A momentum Space Analysis of the Triple Pomeron Vertex in pQCD

We study properties of the momentum space Triple Pomeron Vertex in perturbative QCD. Particular attention is given to the collinear limit where transverse momenta on one side of the vertex are much larger than on the other side. We also comment on the kernels in nonlinear evolution equations.Comment: Minor misprints corrected. To be published in EPJ

Hadrons with Charm and Beauty

By combining potential models and QCD spectral sum rules (QSSR), we discuss the spectroscopy of the $(b\bar c)$ mesons and of the $(bcq)$, $(ccq)$ and $(bbq)$ baryons (${q}\equiv {d}$ or $s$), the decay constant and the (semi)leptonic decay modes of the $B_c$ meson. For the masses, the best predictions come from potential models and read: $M_{B_c} = (6255 \pm 20)$~MeV, $M_{B^*_c} = (6330 \pm 20)$~MeV, $M_{\Lambda(bcu)} = (6.93\pm 0.05)$~GeV, $M_{\Omega(bcs)} = (7.00\pm 0.05)$~GeV, $M_{\Xi^*(ccu)} =(3.63\pm 0.05)$~GeV and $M_{\Xi^*(bbu)} = (10.21\pm 0.05)$~GeV. The decay constant $f_{B_c} = (2.94 \pm 0.21) f_\pi$ is well determined from QSSR and leads to: $\Gamma(B_c \rightarrow \nu_\tau \tau) = (3.0 \pm 0.4)( V_{cb}/0.037 )^2$ $\times 10^{10}$ s$^{-1}$.The uses of the vertex sum rules for the semileptonic decays of the $B_c$ show that the $t$-dependence of the form factors is much stronger than predicted by vector meson dominance. It also predicts the almost equal strength of about 0.30 $\times 10^{10}$ sec$^{-1}$ for the semileptonic rates $B_c$ into $B_s, B^*_s,\eta_c$ and J/$\psi$. Besides these phenomenological results, we also show explicitly how the Wilson coefficients of the $\langle\alpha_s G^2\rangle$ and $\langle G^3\rangle$ gluon condensates already contain the full heavy quark- ($\langle\bar QQ\rangle$) and mixed- ($\langle\bar QGQ\rangle$) condensate contributions in the OPE.}Comment: 32 pages, LaTeX, no changes in the 1994 paper, latex errors corrected in 201

The modulation effect for supersymmetric dark matter detection with asymmetric velocity dispersion

The detection of the theoretically expected dark matter is central to particle physics cosmology. Current fashionable supersymmetric models provide a natural dark matter candidate which is the lightest supersymmetric particle (LSP). Such models combined with fairly well understood physics like the quark substructure of the nucleon and the nuclear form factor and the spin response function of the nucleus, permit the evaluation of the event rate for LSP-nucleus elastic scattering. The thus obtained event rates are, however, very low or even undetectable. So it is imperative to exploit the modulation effect, i.e. the dependence of the event rate on the earth's annual motion. In this review we study such a modulation effect in directional and undirectional experiments. We calculate both the differential and the total rates using symmetric as well as asymmetric velocity distributions. We find that in the symmetric case the modulation amplitude is small, less than 0.07. There exist, however, regions of the phase space and experimental conditions such that the effect can become larger. The inclusion of asymmetry, with a realistic enhanced velocity dispersion in the galactocentric direction, yields the bonus of an enhanced modulation effect, with an amplitude which for certain parameters can become as large as 0.46.Comment: 35 LATEX pages, 7 Tables, 8 PostScript Figures include

New precise determination of the \tau lepton mass at KEDR detector

The status of the experiment on the precise $\tau$ lepton mass measurement running at the VEPP-4M collider with the KEDR detector is reported. The mass value is evaluated from the $\tau^+\tau^-$ cross section behaviour around the production threshold. The preliminary result based on 6.7 pb$^{-1}$ of data is $m_{\tau}=1776.80^{+0.25}_{-0.23} \pm 0.15$ MeV. Using 0.8 pb$^{-1}$ of data collected at the $\psi'$ peak the preliminary result is also obtained: $\Gamma_{ee}B_{\tau\tau}(\psi') = 7.2 \pm 2.1$ eV.Comment: 6 pages, 8 figures; The 9th International Workshop on Tau-Lepton Physics, Tau0

Measurement of RudsR_{\text{uds}} and RR between 3.12 and 3.72 GeV at the KEDR detector

Using the KEDR detector at the VEPP-4M $e^+e^-$ collider, we have measured the values of $R_{\text{uds}}$ and $R$ at seven points of the center-of-mass energy between 3.12 and 3.72 GeV. The total achieved accuracy is about or better than $3.3\%$ at most of energy points with a systematic uncertainty of about $2.1\%$. At the moment it is the most accurate measurement of $R(s)$ in this energy range

Постоянные белки мочи здорового человека в эксперименте с 520-суточной изоляцией

Purpose of the study was to track permanent proteins of urine proteome in the 520-day isolation experiment at the IBMP Ground-Based Test Facility with controlled environmental parameters. Object of the investigation was urine sampled from 6 normal male subjects at the age of 25 to 37 years. Second morning aliquots were gathered during baseline data collection, on days 50, 93, 124, 153, 180, 251, 274, 303, 330, 371, 400 and 427 of isolation, and in 7 days after its completion. Samples were subject to chromatography-mass spectrometry; results were analyzed with the help of bioinformatics resources. The following 7 permanent proteins were observed in urine over the entire length of the investigation: epidermal growth factor, polymer immunoglobulin receptor, plasma serine protease inhibitor, protein AMBP, keratin, type II cytoskeletal 1, collagen alpha-1 (vi) chain, serum albumin